Download The Organellar Genome and Metabolic Potential

The Organellar Genome and Metabolic Potential of the
Hydrogen-Producing Mitochondrion of Nyctotherus ovalis
Rob M. de Graaf,k,1 Guenola Ricard, k,2 Theo A. van Alen,1 Isabel Duarte,2 Bas E. Dutilh,2
Carola Burgtorf,à,1 Jan W. P. Kuiper,1 Georg W. M. van der Staay,§,1 Aloysius G. M. Tielens,3
Martijn A. Huynen,2 and Johannes H. P. Hackstein*,1
1
Abstract
It is generally accepted that hydrogenosomes (hydrogen-producing organelles) evolved from a mitochondrial ancestor.
However, until recently, only indirect evidence for this hypothesis was available. Here, we present the almost complete
genome of the hydrogen-producing mitochondrion of the anaerobic ciliate Nyctotherus ovalis and show that, except for
the notable absence of genes encoding electron transport chain components of Complexes III, IV, and V, it has a gene
content similar to the mitochondrial genomes of aerobic ciliates. Analysis of the genome of the hydrogen-producing
mitochondrion, in combination with that of more than 9,000 genomic DNA and cDNA sequences, allows a preliminary
reconstruction of the organellar metabolism. The sequence data indicate that N. ovalis possesses hydrogen-producing
mitochondria that have a truncated, two step (Complex I and II) electron transport chain that uses fumarate as electron
acceptor. In addition, components of an extensive protein network for the metabolism of amino acids, defense against
oxidative stress, mitochondrial protein synthesis, mitochondrial protein import and processing, and transport of
metabolites across the mitochondrial membrane were identified. Genes for MPV17 and ACN9, two hypothetical proteins
linked to mitochondrial disease in humans, were also found. The inferred metabolism is remarkably similar to the
organellar metabolism of the phylogenetically distant anaerobic Stramenopile Blastocystis. Notably, the Blastocystis
organelle and that of the related flagellate Proteromonas lacertae also lack genes encoding components of Complexes III,
IV, and V. Thus, our data show that the hydrogenosomes of N. ovalis are highly specialized hydrogen-producing
mitochondria.
Key words: hydrogenosome, mitochondrion, horizontal gene transfer, evolution, adaptation, Nyctotherus.
Introduction
Mitochondria are essential organelles in aerobic eukaryotes.
They play a central role in ATP production as well as in
many other cellular processes, such as apoptosis, cellular
proliferation, heme synthesis, steroid synthesis, and Fe/S
cluster assembly (Miller 1995; Lill and Kispal 2000; Scheffler
2001; Duchen 2004). Aerobic mitochondria perform oxidative phosphorylation, that is, they use oxygen as terminal
electron acceptor, oxidizing NADH and FADH2 while producing ATP with the aid of an ATP synthase that exploits
the proton gradient generated by the electron transport
chain. Eukaryotes living under anaerobic circumstances
have evolved a spectrum of organelles that are related
to mitochondria and have names, such as anaerobic mitochondria (both temporarily and permanently anaerobic),
hydrogenosomes, mitosomes, mitochondrial remnants,
modified mitochondria, or mitochondria-like organelles,
reflecting their metabolic peculiarities (Tielens et al.
2002; Barbera et al. 2007; Tachezy and Dolezal 2007;
Tachezy and Smid 2007; Tielens and van Hellemond
2007; Hjort et al. 2010).
Mitochondria that produce ATP via oxidative phosphorylation have a mitochondrial genome, as the protein complexes of proton-pumping electron transport chains
contain essential subunits whose genes were never translocated to the nuclear genome during evolution but always
© The Author(s) 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Mol. Biol. Evol. 28(8):2379–2391. 2011 doi:10.1093/molbev/msr059
Open Access
Advance Access publication March 4, 2011
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Research article
Department of Evolutionary Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, Nijmegen,
The Netherlands
2
Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen
Medical Centre, Nijmegen, The Netherlands
3
Department of Medical Microbiology and Infectious Diseases, Erasmus MC, University Medical Center, Rotterdam, The
Netherlands
Present address: Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland.
àPresent address: Kinzigstr. 3, 78112 St. Georgen, Germany.
§Present address: Kendelweg 2, 47574 Goch, Germany.
kThese authors contributed equally to this work.
*Corresponding author: E-mail: [email protected].
Associate editor: Martin Embley
de Graaf et al. · doi:10.1093/molbev/msr059
remained part of the mitochondrial genome. The canonical
(textbook) mitochondrion uses oxygen as final electron acceptor, but there are also anaerobically functioning mitochondria that use compounds other than oxygen as
final electron acceptor. Most organisms with anaerobic mitochondria use an endogenously produced electron acceptor, such as fumarate (Tielens et al. 2002). Some anaerobic
functioning mitochondria possess a hydrogenase besides
a proton-pumping electron transport chain, and therefore,
they can use protons as final electron acceptors resulting in
the production of hydrogen. These organelles are the socalled hydrogen-producing mitochondria and examples
are found in Nyctotherus ovalis and Blastocystis sp. Hydrogen production in the latter organelles, however, has not
been demonstrated so far (Lantsman et al. 2008; Stechmann et al. 2008). These organelles and those of Proteromonas, for which currently nothing is known about
hydrogen production, are considered to be bona fide mitochondria as they use a proton-pumping electron transport chain and hence possess a mitochondrial genome
encoding essential parts of it (Perez-Brocal et al. 2010).
Hydrogenosomes are mitochondrion-related organelles
that, by definition, produce ATP and hydrogen using protons as electron acceptors. In contrast to hydrogen-producing mitochondria, hydrogenosomes have no
mitochondrial genome, do not possess a proton-pumping
electron transport chain, and produce ATP exclusively via
substrate-level phosphorylation. Hydrogenosomes are
found in various unrelated anaerobic eukaryotes, such as
anaerobic flagellates, chytridiomycete fungi, and several anaerobic ciliates (Hackstein et al. 2001; Embley et al. 2003;
Embley and Martin 2006; Hackstein, Baker, et al. 2008;
Hackstein, de Graaf, et al. 2008; de Graaf, Duarte, et al.
2009).
Mitosomes produce neither hydrogen nor ATP, but in
general have retained components of a Fe/S cluster synthesizing machinery, which is now believed to be the reason
why mitochondria, hydrogenosomes, and mitosomes are
essential to eukaryotic life (Henze and Martin 2003; Yarlett
2004; Tachezy and Dolezal 2007; Goldberg et al. 2008). Even
for Entamoeba (Aguilera et al. 2008) that has been regarded
as the potential exception to this pattern, evidence is now
mounting that its mitosomes also assemble Fe/S clusters
(Maralikova et al. 2010), although Mi-ichi et al. (2009) could
not confirm this finding using a proteomics approach. All
eukaryotic organisms studied so far in sufficient molecular
and ultrastructural detail appear to possess either mitochondria, hydrogenosomes or mitosomes. Furthermore,
no species has been found that contains both mitochondria and mitosomes or mitochondria and hydrogenosomes,
supporting the hypothesis that these organelles have
a common origin. Despite this common origin, mitochondria, hydrogenosomes, and mitosomes are very diverse: not
only in the size of their proteomes but also by virtue of their
protein composition and function (Gabaldón and Huynen
2004). However, classical aerobic mitochondria share two
characteristics: They have retained a genome, allowing unambiguous documentation of their descent from an a-pro2380
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teobacterium by phylogenetic analyses (Lang et al. 1999),
and they contain an electron transport chain with Complexes I through IV as well as an ATPase (Complex V).
In the evolution of mitochondria, hydrogenosomes and
mitosomes from an a-proteobacterium, a large number
of genes and proteins have been lost, gained from various
sources, or retargeted to other organelles, thus shaping the
huge diversity of current mitochondria and their homologs
in various species (Gabaldón and Huynen 2004). Within the
evolutionary ‘‘gap’’ between species with genome-containing
mitochondria and species with mitosomes or hydrogenosomes (both lacking an organellar genome), the species
N. ovalis provides an interesting link. Nyctotherus ovalis is
an anaerobic ciliate that lives in the hindgut of various
cockroach species. It has numerous hydrogen-producing
organelles that are intimately associated with endosymbiotic methane–producing archaea that use the hydrogen
produced by the organelles. Despite the hydrogenosomal
metabolism of this organelle, we have found that it actually
has a genome (Akhmanova, Voncken, van Alen, et al. 1998),
that similar to mitochondrial genomes (Gray 2005), encodes elements of an electron transport chain (Boxma et al.
2005). Phylogenetic analyses of the organellar genome of N.
ovalis showed that it has evolved from the genome of an
aerobic mitochondrion of a ciliate ancestor (Boxma et al.
2005), providing direct evidence that hydrogenosomes can
evolve from mitochondria. This blurs the distinction between mitochondria and hydrogenosomes. A comparable
situation is found in the mitochondrion-like organelles of
the phylogenetically only distantly related Stramenopile
Blastocystis that is clearly distinct from the ciliates, which
belong to the Alveolata. Blastocystis also retains a mitochondrial genome and parts of an electron transport chain
(Perez-Brocal and Clark 2008; Stechmann et al. 2008;
Wawrzyniak et al. 2008). Because this organelle also hosts
a [FeFe] hydrogenase, it can be regarded as a hydrogenosome with genome, although thus far no hydrogenase
activity has been demonstrated in this species (Lantsman
et al. 2008; Stechmann et al. 2008).
Also for other (genome-less) types of hydrogen-producing
organelles evidence is accumulating that they have evolved
from mitochondria (Embley and Martin 2006). In the absence of an organellar genome, the evidence for homology
is based on phylogenetic analyses of proteomes. Based on
phylogenetic analyses of Complex I subunits, the hydrogenosomes of the parabasalid Trichomonas are inferred to
share a common ancestor with mitochondria (Hrdý et al.
2004). Also, the hydrogenosomes of anaerobic chytrids
seem to have evolved from the mitochondria of their aerobic
ancestors (Akhmanova, Voncken, Harhangi, et al. 1998). It is
therefore likely that hydrogenosomes arose repeatedly by
evolutionary tinkering as an adaptation to the particular requirements of hosts, which thrive in rather different anaerobic environments.
Here, we describe the isolation and sequence analysis of
the major part (41,666 bp) of the organellar genome of
N. ovalis and compare it with the mitochondrial genomes
of the ciliates Paramecium aurelia, Tetrahymena spp., and
Hydrogen-Producing Mitochondria of Nyctotherus · doi:10.1093/molbev/msr059
Euplotes minuta as well as with the mitochondrial/hydrogenosomal genome of the Stramenopiles Blastocystis sp. and
Proteromonas lacertae. We show that the organellar genome of N. ovalis is a typical ciliate mitochondrial genome
except for the obvious absence of genes encoding components of Complexes III, IV, and V. The lack of evidence for
the presence of these genes in N. ovalis coincides with the
definitive absence of these genes from the organellar genomes of Blastocystis sp. and P. lacertae (Perez-Brocal
and Clark 2008; Stechmann et al. 2008; Wawrzyniak
et al. 2008; Perez-Brocal et al. 2010). By comparing the hydrogenosomal proteins and metabolic pathways of N. ovalis with the various hydrogenosomes, mitosomes,
mitochondria-like organelles, and mitochondria, we show
that the N. ovalis organelle is as complex as some mitochondria.
Materials and Methods
Cell Isolation
Nyctotherus ovalis cells were isolated from the hindgut
of the cockroach Blaberus sp. var. Amsterdam. Total
DNA was isolated by dissolving living cells in 8 M guanidinium chloride and separation on a hydroxyapatite
column using standard procedures (de Graaf, van Alen,
et al. 2009).
The organellar DNA was isolated from the total DNA by
pulsed field gel electrophoresis. This organellar DNA was
used for a partial Sau 3A I digest and cloning in the
Bam H1 restriction site from the vector pUC18c. From this
library, 500 clones were sequenced in both directions and
additional 500 in one direction. Less than 3% of these
clones contained a mitochondrial sequence. Finally, larger
pieces of the organellar genome were reconstructed, and
the gaps were filled by long range polymerase chain reaction (PCR) with specific primers resulting in a contig of
41,666 bp representing the major part of the organellar genome (accession number: GU057832). Due to the very limited amount of DNA available, we did not attempt to
sequence the chromosome ends.
Identification of N. ovalis sequences likely encoding
hydrogenosomal proteins
Nyctotherus ovalis sequences translated in the six frames
were compared with the mitochondrial proteomes of human, yeast, rat, and Tetrahymena. The mitochondrial proteomes were retrieved from the mitoproteome (http://
www.mitoproteome.org), the SGD database (Cherry
et al. 1998), the supplementary material from Sickmann
et al. (2003), from Forner et al. (2006), and from Smith
et al. (2007). The accession numbers of the N. ovalis sequences are displayed in supplementary table S1, Supplementary Material online. Nyctotherus ovalis sequences with
a reciprocal best hit (BH) with one of the mitochondrial
proteins (Smith Waterman, E , 0.01) were selected for
further phylogenetic analysis to confirm orthology
with a mitochondrial protein. Each nucleotide sequence
selected was compared by the SWX algorithm with the
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165 complete proteomes, and the first 100 hits were kept
and used together with the N. ovalis sequence to produce
a multiple alignment with Muscle v3.7 (default parameters;
Edgar 2004). Positions of the alignments that did not
contain gaps were automatically selected, and a tree was
subsequently derived using PhyML (Guindon and Gascuel
2003) using the JTT model and an estimated number
of invariable sites with four substitution rate categories.
Hundred bootstraps were performed.
Phylogenetic Position
To determine the phylogenetic position of the N. ovalis hydrogen-producing mitochondrion, we composed a concatenated alignment phylogeny of the Complex I proteins that
tend to be encoded on the mitochondrial genome (nad1,
nad2, nad3, nad4, nad4L, nad5, and nad6), using the
gamma-proteobacterium Escherichia coli and the a-proteobacterium Rickettsia prowazekii as outgroups. The sequences in each protein family were first aligned with
Muscle (Edgar 2004). We then concatenated the alignment
blocks, where gaps were inserted in the rare cases that
a protein was missing from a certain species (e.g., nad6
from N. ovalis). Given the length of the alignment, we
opted to compute a bootstrapped Maximum Likelihood
(ML) phylogeny. For this, we first selected the best-fit
model of amino acid replacement, using the Akaike information criterion (AIC) as goodness of fit measure, as implemented in ProtTest (version 2.2) (Abascal et al. 2005).
Accordingly, a ML phylogeny, 100 times bootstrapped,
was computed with PhyML (version 3.0.1) (Guindon and
Gascuel 2003) using the previously selected LG model of
amino acids substitution, with a discrete gamma distribution approximated by 4 rate-categories (þ4G), estimated
proportion of invariable sites (þI), and observed amino
acid frequencies (þF).
Identification of Sequences Derived From HGT
In order to identify horizontal gene transfers (HGTs) present in the N. ovalis genome, we built phylogenies for all the
proteins that had BHs with noneukaryotic proteins in
a Blast search. We carefully selected the homologous data
set from the 250 BHs in the nonredundant database, supplemented with ten phylogenetically well distributed, best
Blast hits from eukaryotes only, aligned the sequences using
ClustalW (version 2.0.10) (Thompson et al. 1994), and subsequently pruned the alignment for redundancy. Each
alignment was then submitted to ProtTest (version 2.4)
(Abascal et al. 2005) in order to statistically infer the
best-fit model of amino acid substitution, according to
the AIC. Finally, a ML phylogeny was computed with
PhyML (version 3.0.1) (Guindon and Gascuel 2003) employing the previously chosen model and bootstrapped
100 times.
Subcellular Localization
Mitop2 (Andreoli et al. 2004) was used when the N-terminal
part of the N. ovalis sequence was present to examine the
presence of a putative Mitochondrial Import Signal.
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de Graaf et al. · doi:10.1093/molbev/msr059
N. ovalis cDNA and gDNA
Nyctotherus ovalis cDNA and genomic DNA (gDNA) libraries were constructed as described earlier (Ricard et al.
2008). The macronuclear gDNA clones were obtained after
amplification of total ciliate DNA with telomere primers. In
general, only gDNA clones with at least one telomere
were used in order exclude contamination by bacterial
sequences.
Results and Discussion
Gene Content and Structure of the Organellar
Genome of N. ovalis
In 2005, Boxma et al. showed that a 14 Kb fragment of the
organellar genome of the anaerobic hydrogen-producing
ciliate N. ovalis that had been isolated from the cockroach
Blaberus sp. var. Amsterdam was of mitochondrial origin
(Boxma et al. 2005). Here, we describe an analysis of the
nearly complete organellar genome. Pulsed field gel electrophoresis followed by Southern blot hybridization with two
different 32P-labeled probes, nad7 and 12S (rns), indicated
that the size of the organellar genome of N. ovalis exceeds
48 kb (supplementary fig. S1, Supplementary Material
online).
Mitochondrial genes present in the organellar library
were identified by Blast analysis (Altschul et al. 1997),
and in the case of nonoverlap, subjected to long-range
PCR to fill the gaps between the fragments. Using this
approach, the major part of this genome (41,666 bp)
has been sequenced and reconstructed as a single contig.
This part of the organellar genome contains an almost
complete set of genes found in the mitochondrial genomes
of other ciliates (table 1, fig. 1). In addition, we found
a stretch larger than 11 kb that possesses seven open
reading frames (orfs) with no significant sequence similarity
to any known genes.
The organellar genome contains nine genes encoding elements of mitochondrial Complex I: nad1, nad2, nad3,
nad4, nad4L, nad5, nad7, nad9, and nad10. This set of genes
is also found in other ciliates (table 1). The nad1 gene is split
in the mitochondrial genomes of all ciliates studied so far
into a larger (nad1a) and a smaller (nad1b) part (de Graaf,
van Alen, et al. 2009). In N. ovalis, we could identify the
larger nad1a gene piece while the nad1b gene is likely encoded by orf224 (fig. 1). The other Complex I genes have
a similar size as in the Tetrahymena species and P. aurelia.
This is in contrast to the situation in E. minuta and E. crassus where many Complex I genes have extended 5# ends
(de Graaf, van Alen, et al. 2009). The nad4 gene of N. ovalis
is a bit shorter than in other ciliates, and it is very likely that
orf110 represents an additional part of the nad4 gene
(fig. 1).
Phylogenetic analysis of seven concatenated Complex I
genes reveals that the Complex I of N. ovalis is closely related
to that of E. crassus and E. minuta and that it clusters together with the Complex I genes of Tetrahymena and Paramecium (fig. 2), whereas it is—as expected—only distantly
related to Complex I of Blastocystis and Proteromonas.
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Organellar genes encoding components of Complexes
III, IV, or V could not be identified. These genes are consistently present in the mitochondrial genomes of the aerobic ciliates P. aurelia, Tetrahymena spp., and E. minuta
that have a size around 48 kb (table 1; de Graaf, van Alen,
et al. 2009). Moreover, as a rule, all mitochondrial genomes
sequenced to date host genes belonging to Complexes III,
IV, and V (Burger et al. 2003). The loss of genes encoding
Complexes III, IV, and V from the mitochondrial genome
has been regarded as indicative of a loss from the species rather
than a transfer to the nuclear genome in the mitochondrionlike organelles of Blastocystis (Perez-Brocal and Clark 2008;
Stechmann et al. 2008; Wawrzyniak et al. 2008). The absence
of Complex III and IV genes in the organellar genome of N.
ovalis is consistent with the observation that activity of
Complexes III and IV could not be observed in inhibitor studies (Boxma et al. 2005).
A total of 15 unique orfs were identified, seven of which
form a cluster with a total length greater than 11 kb with
no significant sequence similarity to any known genes. No
open reading frames with sequence similarity to orfs of Tetrahymena spp. P. aurelia, and E. minuta were identified. We
detected three transfer RNA genes in the organellar genome (trnF, trnY, and trnW), similar to the mitochondrial
genome of P. aurelia in which four transfer RNA (tRNA)
genes were found (Pritchard et al. 1990). It is possible that
additional tRNA genes are present in the missing part of
the organellar genome. A large complex repeat region is
present in the organellar genome between orf479 and
ssu/rns genes. This region contains 12 repeats of 34 nt followed by a (not perfectly) duplicated stretch of around
400 bp (supplementary fig. S2, Supplementary Material online). It is unlikely that this repeat region plays the same role
in initiation of transcription as suggested for the mitochondrial genome of the E. minuta and E. crassus (de Graaf, van
Alen, et al. 2009) because all genes found on the 41,666 bp
part of the organellar genome of N. ovalis are oriented in
the same direction, both before and after the repeat. In
E. minuta and E. crassus, the repeat separates the open
reading frames into two blocks, each having a single direction of transcription, from the repeat toward the ends of
the chromosome.
We identified seven ribosomal proteins in the organellar
genome of N. ovalis. This seems to be an average number
for ciliates because nine ribosomal proteins were found in
Tetrahymena spp., seven in P. aurelia, and six in E. minuta
(de Graaf, van Alen, et al. 2009). Three of these ribosomal
proteins were found in all four ciliate species with sequenced mitochondrial genomes: Rps12, Rpl2, and
Rpl14. In contrast, the rps8 gene is unique for N. ovalis
(table 1). Notably, as in E. minuta, all organellar ribosomal
protein genes of N. ovalis have an N-terminal extension that
is similar to a mitochondrial targeting signal (not shown, cf.
Ueda et al. 2008; de Graaf, van Alen, et al. 2009). The overall
A-T content of the organellar genome of N. ovalis (58.5%) is
identical to the A-T content of the mitochondrial genome
of P. aurelia. The organellar genes are not tightly packed,
and intergenic spacers represent 11.7% of the genome. In
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Table 1. Various Genes Encoded by the Organellar Genomes of Nyctotherus ovalis (Nov), Blastocystis sp. (Blas), Proteromonas lacertae (Pro),
Euplotes minuta (Emi), and Plasmodium falciparum (Pfa).
Gene
nad1
nad2
nad3
nad4
nad4L
nad5
nad6
nad7
nad9
nad10
nad11
rnl
rns
cob
cox1
cox2
cox3
atp9
ccmF/yejR
rps2
rps3
rps4
rps7
rps8
rps10
rps11
Nov
Blas
Pro
Emi
n
n
n
n
n
n
h
n
n
n
h
n
n
h
h
h
h
h
h
h
h
h
h
n
h
h
n
n
n
n
n
n
n
n
n
h
n
n
n
h
h
h
h
h
h
h
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
h
n
n
n
h
h
h
h
h
h
n
h
n
h
n
n
h
n
n
n
n
n
n
h
n
n
n
h
n
n
n
n
n
h
n
n
h
n
h
h
h
h
h
Pfa
h
h
h
h
h
h
h
h
h
h
h
n
n
n
n
h
n
h
h
h
h
h
h
h
h
h
Gene
rps12
rps13
rps14
rps19
rpl2
rpl5
rpl6
rpl14
rpl16
trnA
trnC
trnD
trnE
trnF
trnH
trnI
trnK
trnL
trnM
trnN
trnP
trnQ
trnS
trnV
trnW
trnY
Nov
Blas
Pro
Emi
n
h
n
h
n
h
n
n
h
h
h
h
h
n
h
h
h
h
h
h
h
h
h
h
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
h
h
h
n
n
n
h
n
n
n
n
n
n
n
h
h
n
n
n
h
n
n
h
n
n
n
n
n
n
n
n
n
h
h
h
n
h
n
n
n
h
h
h
n
n
n
h
h
h
n
h
h
n
h
h
n
n
Pfa
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
NOTE.—n: present in the organellar genome; h: absent in the organellar genome.
contrast, the genomes of the other ciliates contain no more
than about 4% of intergenic spacers. The A-T content of
these spacers is almost identical to the overall A-T content:
57.8%. Spacer lengths vary from 0 to 2,159 bp with an average size of 320 bp. In six cases, genes have an overlap,
ranging from 3–61 bp. The 11 kb fragment that harbors
the cluster of seven orfs with no homology to other known
genes contains large open reading frames with small intergenic spacers ranging from 3 to 9 bp. These seven genes
do not appear to be the remains of genes encoding components of Complex III, Complex IV, and Complex V.
Neither Blast searches against mitochondrial encoded proteins nor profile-based searches against domain databases
(PFAM, SMART) produced hits, even at an insignificant
E value cutoff of 10. Because none of the proteins has
a pI . 10, it is also unlikely that they are ribosomal proteins.
Thus, the sequenced fragment of the organellar genome of
N. ovalis exhibits all characteristics of a ciliate mitochondrial
genome, except for the absence of genes encoding components of the mitochondrial Complexes III, IV, and V.
Nuclear Encoded Organellar Proteins
As in other mitochondria, most of the organellar proteins
in N.ovalis are encoded by the nuclear genome, synthesized in the cytoplasm, and imported into the organelle.
The signal to direct these proteins into the hydrogen-producing mitochondrion resembles mitochondrial targeting
signals (Boxma et al. 2005) and can be an indication that
a certain protein is targeted to the organelle. Furthermore,
because mitochondrial proteins tend to preserve their cellular location in evolution (Calvo et al. 2006; Szklarczyk
and Huynen 2009), the orthology of nuclear encoded mitochondrial proteins from species like Homo sapiens, Saccharomyces cerevisiae, and T. thermophila with N. ovalis
proteins can be used to predict hydrogenosomal proteins.
Here, we describe in more detail nuclear encoded proteins
that are probably targeted to the hydrogen-producing mitochondrion of N. ovalis.
After our initial analysis (Boxma et al. 2005), we have
obtained ;4,500 additional N. ovalis gDNA and cDNA
sequences (Ricard et al. 2008). To identify new proteins
involved in the hydrogenosomal metabolism, we first performed a Bidirectional BH analysis. Thousand nine hundred
and fourteen nonredundant cDNAs and 2,841 nonredundant gDNAs from N. ovalis were compared with the mitochondrial proteomes of human, yeast, and rat. This allowed
us to identify orthologs of mitochondrial proteins for which
161 phylogenetic trees were constructed with PhyML and
subsequently analyzed by hand to determine orthology relations (not shown). This phylogenetic analysis allowed us
to predict a number of new hydrogenosomal proteins as
well as to provide stronger evidence for some that had been
predicted previously (Boxma et al. 2005). From the phylogenetic trees, we observed that most of the N. ovalis sequences with close relatives among the mitochondrial
proteins also cluster with their counterparts from the aerobic ciliates T. thermophila and P. tetraurelia. This indicates
their recent descent from proteins functioning in aerobic
mitochondria (data not shown). Furthermore, to compare
the metabolic complexity of the N. ovalis hydrogenproducing mitochondrion with that of mitochondria, genome-less hydrogenosomes, and mitosomes, we analyzed
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FIG. 1. Organellar gene maps of Nyctotherus ovalis and Euplotes minuta. Red: Complex I genes, blue: rRNA genes, green: ribosomal proteins,
yellow: Complex III and IV genes, gray: unidentified open reading frames, pink: repeat region, dark gray: atp9 gene, and white: intergenic spacers.
Capital letters indicate the various tRNA genes. Arrows: direction of transcription.
the distribution of N. ovalis proteins with sequence
similarity to organellar proteins in other species. These
were the mitochondria-possessing species Homo, Saccharomyces, Reclinomonas, Plasmodium, Tetrahymena, and
Paramecium; the species possessing a mitosome Cryptosporidium, Encephalitozoon, Giardia, and Entamoeba;
and the species with a hydrogenosome Piromyces, Trichomonas, Psalteriomonas, and Blastocystis (supplementary
table S1, Supplementary Material online). Here, we describe some of the proteins and their roles. Figure 3 shows
how they might function together, shaping the organellar
metabolism:
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Pyruvate Dehydrogenase
The PDH complex converts pyruvate and CoA into acetylCoA while reducing NADþ to NADH. In addition to the
three subunits, which have already been described (E1a,
E1b, and E2), we found a fourth subunit, a dihydrolipoyl
dehydrogenase, also known as subunit E3, thereby
completing the complex. The presence of a complete
PDH complex contrasts strongly with the pyruvate catabolizing enzymes in other hydrogenosomes. Although the
genome analysis of Blastocystis revealed a PDH and a pyruvate:ferredoxin oxidoreductase (PFO) (Stechmann et al.
2008), Lantsman et al. (2008) measured pyruvate:NADPþ
Hydrogen-Producing Mitochondria of Nyctotherus · doi:10.1093/molbev/msr059
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among the gDNA sequences of N.ovalis, we did encounter
a malate dehydrogenase that is orthologous to cytosolic malate dehydrogenases from other species and was therefore
considered to be also cytoplasmic in N. ovalis as well.
FIG. 2. ML phylogeny of the Nyctotherus ovalis hydrogen-producing
mitochondrion, based on a concatenated alignment of seven
mitochondrial Complex I encoded proteins (Nad1, Nad2, Nad3,
Nad4, Nad4L, Nad5, and Nad6). Only bootstrap values of 50% and
higher are indicated.
oxidoreductase activity. Thus, the situation in Blastocystis is
unclear. In Trichomonas pyruvate is decarboxylated by PFO
(Hrdý et al. 2007). In contrast, in anaerobic chytridiomycete
fungi (Piromyces sp. E2 and Neocallimastix sp. L2), pyruvate
is catabolyzed by pyruvate-formate lyase (PFL) and not by
PFO (Akhmanova et al. 1999). Furthermore, a large cDNA
library of Piromyces constructed by the Department of Energy (DOE) Joint Genome Institute does not contain a single
PFO but many PFL sequences (data not shown). The acetylCoA generated in N. ovalis by PDH is further metabolized
by an acetate:succinate CoA-transferase (ASCT) belonging
to the subfamily 1A with sequence similarity to the ASCT
from Trypanosoma brucei (Tielens et al. 2010). Finally, ATP
is generated by the action of succinyl-CoA synthetase
(SCS), an enzyme that is also part of the tricarboxylic acid
(TCA) cycle, but this cycle is not operative in N. ovalis
(fig. 3). Also in Blastocystis and Trichomonas acetyl-CoA
is metabolized to acetate by the cyclic action of ASCT
and SCS. However, Blastocystis contains genes for ASCTs
of the subfamilies 1B and 1C, and Trichomonas contains
a gene for an ASCT of the subfamily 1C that does not
exhibit sequence similarity to the subfamily 1A ASCT of
N. ovalis (supplementary table S1, Supplementary Material
online; Stechmann et al. 2008; Tielens et al. 2010).
TCA Cycle
Based on experiments with 14C labeled glucose (Boxma et al.
2005), the TCA cycle in N. ovalis is not complete, using only
the malate–fumarate–succinate part in a reductive direction
(fig. 3). Thus far, however, we have only found succinyl-CoA
synthetase a and b subunits (the same enzyme as mentioned above) as well as the a and b subunit of succinate
dehydrogenase (SDH)/fumarate reductase. These enzymes
are also present in the mitochondrion-like organelle of Blastocystis in which succinyl-CoA synthetase, SDH, fumarate hydratase, and malate dehydrogenase have been identified
(supplementary table S1, Supplementary Material online)
(Stechmann et al. 2008; Wawrzyniak et al. 2008). These enzymes are universal in species with mitochondria. Note that
Electron Transport Chain
Complex I (NADH-quinone oxidoreductase) consists of 14
subunits in eubacteria and of 33–45 subunits in mitochondria
(Friedrich and Böttcher 2004; Gabaldon et al. 2005). We found
12 subunits of Complex I in N. ovalis (Nad1, Nad2, Nad3,
Nad4, Nad4L, Nad5, Nad7, Nad9, Nad10, 24kDa*, 51kDa*,
and 75kDa*), among them three that are encoded by the nuclear genome (marked by an asterisk); the others are encoded
by the hydrogenosomal genome (fig. 1). All the N. ovalis Complex I proteins are part of the 14 proteins that compose the
core bacterial Complex I. Also in Blastocystis, ten Complex I
genes have been identified that are located on the organelle
genome, and an additional six that are nuclear encoded (Perez-Brocal and Clark 2008; Stechmann et al. 2008; Wawrzyniak
et al. 2008). In Proteromonas, ten Complex I proteins are encoded by the organellar genome (Perez-Brocal et al. 2010).
Of complex II, we detected SDHa and SDHb. In aerobic
mitochondria, the four subunits of Complex II catalyze the
oxidation of succinate to fumarate and the reduction of ubiquinone to ubiquinol. In N. ovalis, SDHa and SDHb have been
proposed to function as fumarate reductase, reversing the
reaction, and allowing the oxidation of quinols (rhodoquinol)
(Boxma et al. 2005). In Blastocystis, all four components
of Complex II have been identified (Stechmann et al. 2008).
Previous biochemical analyses have failed to detect the
presence of Complex III/IV activity in the N. ovalis hydrogenproducing mitochondrion (Boxma et al. 2005), and accordingly, the analysis of its genome did not provide any
evidence for the presence of Complex III/IV genes (fig.
1). Surprisingly, however, we did identify a nuclear-encoded
homolog of cytochrome c1, an electron transporter related
to Complexes III and IV. We sequenced the complete genesized-piece as well as the complete cDNA, confirming that
the cytochrome c protein is transcribed. However, the
cDNA appears to contain a stop codon (TGA), albeit
one that is very rarely used. Therefore, with our current
knowledge, we cannot exclude (Ricard et al. 2008) that
the cDNA is translated notwithstanding the presence of
a potential stop codon. Accordingly, an alignment of the
cytochrome c sequence with homologs from various species reveals that the sequence is highly conserved but that
the cysteines that normally hold the heme in cytochrome
c1 are not present in the N. ovalis sequence, probably rendering the protein nonfunctional in electron transfer (supplementary fig. S3, Supplementary Material online).
Therefore, if the gene is translated, it should exhibit an
alternative function. This is the first report of a cytochrome
c1 that has lost the two crucial cysteines as well as the
histidine directly following the second cysteine. Only
some Trypanosoma, Leishmania, and Euglena species miss
the first cysteine, whereas the second cysteine and the
histidine are universally conserved among species (Priest
and Hajduk 1992).
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FIG. 3. Tentative reconstruction of the metabolism of the hydrogen-producing mitochondria of Nyctotherus ovalis. The metabolism is based on
proteins that are orthologous to mitochondrial proteins (Methods) and proteins derived from HGT that are likely to have an organellar location
based on their metabolic function. It includes the results of metabolic experiments described in Boxma et al. (2005). In red: metabolism linked to
NADH (green) oxidation and electron transfer as well as solute carriers. In yellow are the proteins that seem to have been acquired by HGT. In blue:
glycine metabolism. In orange: intermediate metabolism. Dotted arrows stand for metabolic steps we assume to be present based on biological
experiments, broken arrows for the inferred metabolism for which we did not find the gene yet. AAC: ADP/ATP carrier; ACS: acetyl-CoA synthetase,
AMP-forming (EC: 6.2.1.1); ALT: alanine amino transferase; ASCT: acetate:succinate CoA transferase; Cyt c1: cytochrome c1; CbS: cystathione b
synthase; EfTu: elongation factor Tu; ETF: electron transfer flavoprotein; Fe-hyd: FeFe hydrogenase; FRD/SDH: fumarate reductase/succinate
dehydrogenase; GDH: glutamate dehydrogenase; GLO1: glyoxalase I; MCF Pet 8: mitochondrial carrier family; MDH: malate dehydrogenase; ME:
malic enzyme; Met tRNA ft: methionyl-tRNA formyltransferase; MMM: methyl malonyl CoA mutase; MOC: malate:oxoglutarate carrier; NADH-DH:
NADH :quinone oxidoreductase; PCC: propionyl CoA carboxylase; PDH: pyruvate dehydrogenase; RQ: rhodoquinone; SCS: succinyl-CoA synthetase;
SHMT: serine hydroxymethyl transferase; *includes several enzymes involved in branched-chain amino acid metabolism.
We did not find genes encoding Complex V proteins in
N. ovalis that could use the proton motive force generated
by Complex I (Boxma et al. 2005). An organelle-encoded
component of Complex V could not be identified in Blastocystis and Proteromonas either (Perez-Brocal and Clark
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2008; Stechmann et al. 2008; Wawrzyniak et al. 2008; Perez-Brocal et al. 2010).
Another interesting protein involved in electron transfer
is the a subunit of the electron transfer flavoprotein (ETF).
ETF is an electron acceptor for various mitochondrial
Hydrogen-Producing Mitochondria of Nyctotherus · doi:10.1093/molbev/msr059
dehydrogenases, for example, dehydrogenases that oxidize
amino acids. It transfers electrons to the electron transport
chain via an ETF-ubiquinone oxidoreductase. The presence
of ETF underscores the importance of the N. ovalis electron
transport chain in its mitochondrial catabolism.
With the notable exception of Blastocystis and Proteromonas, complete functional Complexes I and II appear to
be absent from hydrogenosomal species, such as Trichomonas, which has only retained two subunits of Complex I
(the 24 and the 51 kD subunits) (Hrdý et al. 2004; Carlton
et al. 2007; Stechmann et al. 2008). The electron transport
chain is also absent from all mitosomal species. All these
species lack a mitochondrial genome, whereas N. ovalis,
Blastocystis, and Proteromonas possess elements of the
electron transport chain as well as a mitochondrial genome. This supports the conclusion that all mitochondrial
genomes carry genes encoding proteins of the electron
transport chain (Burger et al. 2003), and likewise, that all
organisms with an electron transport chain have a mitochondrial genome.
Propionate Metabolism
The predicted mitochondrial proteome contains a propionyl CoA carboxylase (EC: 6.4.1.3), that catalyzes the carboxylation reaction of propionyl CoA to D-methylmalonyl
CoA, and a methylmalonyl CoA mutase (also found in Blastocystis, Stechmann et al. 2008) that converts methylmalonyl CoA to succinyl CoA. Both propionyl CoA
carboxylase and methylmalonyl CoA mutase are absent
from Paramecium and Tetrahymena (supplementary table
S1, Supplementary Material online), indicating that the
hydrogen-producing mitochondrion of N. ovalis does
not just possess a subset of proteins present in aerobic
mitochondria.
Fatty Acid Metabolism
In the fatty acid activation pathway, we detected a longchain-fatty acid-CoA ligase, which catalyzes the reversible
reaction:
ATP þ a long-chain carboxylic acid þ CoA ,5. AMP
þ diphosphate þ acyl-CoA.
This enzyme is present in all the 11 organisms in
supplementary table S1, Supplementary Material online
for which complete genomes are available, regardless
of whether these organisms possess mitochondria, hydrogenosomes, or mitosomes. In contrast, the glycerol
kinase is only present in the seven organisms that possess
mitochondria or hydrogenosomes. Organisms with
mitosomes lack glycerol kinase, with the exception of
G. lamblia (supplementary table S1, Supplementary
Material online).
Amino Acid Metabolism
Besides the glycine cleavage system (GCS, see below), we
found several other enzymes involved in the metabolism
of amino acids and specifically in the degradation of valine, methionine, and isoleucine. These were a branchedchain aminotransferase (EC: 2.6.1.42), the 2-oxoisovalerate
dehydrogenase a and b (EC: 1.2.4.4) subunits, and
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a branched-chain a-keto acid dihydrolipoyl acyltransferase (EC: 2.3.1.-). The organelle of Blastocystis also contains
enzymes involved in valine, leucine, and isoleucine metabolism (Stechmann et al. 2008). In addition, we identified a
glutamate dehydrogenase and a cystathionine b-synthase.
In general, amino acid metabolism appears much reduced
in mitosomes and genome-less hydrogenosomes relative
to mitochondria (supplementary table S1, Supplementary
Material online).
GCS
The dihydrolipoyl dehydrogenase of PDH is also involved in
the GCS that catalyzes the reversible reaction:
glycine þ tetrahydrofolate þ NADþ 5 5,10-methylenetetrahydrofolate þ NH3 þ CO2 þ NADH þ Hþ
It provides the 5,10-methylene-tetrahydrofolate that is
required for nucleotide synthesis. Of the GCS, we detected:
the H-protein, a lipoic acid containing protein that carries
the aminomethyl moiety of glycine that is bound to the
sulfhydryl group by an S-C bond; the T-protein, which degrades reversibly the aminomethyl moiety attached to the
H-protein to methylene-tetrahydrofolate and ammonia,
using tetrahydrofolate in the process; and the L-protein,
a dihydrolipoamide dehydrogenase that catalyzes the oxidation of the dihydrolipoyl group of the H-protein. We did
not detect the substrate determining pyridoxal phosphatecontaining protein (P-protein) of this complex, casting
some doubt on whether the system actually uses Glycine.
Nevertheless, we did find other components linked to this
pathway: a cystathionine b-synthase, a serine hydroxymethyl transferase that converts serine to glycine, and
a 5,10-methenyltetrahydrofolate synthetase. Interestingly,
two proteins of the GCS have also been identified in the
hydrogenosome of Trichomonas (L- and H-proteins) (Carlton et al. 2007), whereas the L, T, and H proteins also appear to be present in the genome of the minimal
mitochondria-containing Plasmodium falciparum (supplementary table S1, Supplementary Material online). The
mitochondrion-like organelle of Blastocystis contains all four
components of the GCS, including the P-protein (Stechmann
et al. 2008). The GCS is completely absent from mitosomeharboring species, such as Encephalitozoon cuniculi, E. histolytica, G. lamblia, and C. parvum.
Fe/S Cluster Synthesis
The only characteristic known so far that is shared by
all mitochondria, mitosomes, and hydrogenosomes is the
synthesis of Fe/S clusters (Lill and Mühlenhoff 2005;
Tachezy and Dolezal 2007). Unfortunately, we found only
a mitochondrial type I [2Fe2S] ferredoxin, which might
provide reducing equivalents for Fe/S cluster synthesis as
well as for other processes. Furthermore, we found the
mitochondrial Hsp70, which has a role in Fe/S assembly
and in other processes, such as protein import and folding.
Although we have no reason to doubt their presence, we
did not detect genes specific for Fe/S cluster assembly like
Isu1/2, Nfs1, Isd11, Isa1/2, or frataxin and therefore have no
specific evidence for canonical mitochondrial Fe/S cluster
synthesis in N. ovalis.
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Mitochondrial Protein Synthesis and Turnover
We found eight mitochondrial ribosomal proteins in N.
ovalis: Rps 8, Rps 12, Rps 14; Rpl 2, Rpl 6, Rpl 14, Rpl 11,
Rpl 15, and Rpl 20; the latter three are nuclear encoded.
In agreement with Smits and coworkers (Smits et al.
2007), we did not find these mitochondrial ribosomal proteins in species lacking a mitochondrial genome, such as G.
lamblia, E.cuniculi, Ent. histolytica, C. parvum, or T. vaginalis.
Notably, 16 ribosomal proteins are encoded in the genome of the mitochondrion-like organelle of Blastocystis
(Perez-Brocal and Clark 2008; Stechmann et al. 2008; Wawrzyniak et al. 2008). In N. ovalis, we found, besides the ribosomal proteins, FtsJ (MRM2), a well-conserved heat shock
protein that is responsible for methylating 23S (rnl) rRNA.
Mitochondrial Carriers
We detected three mitochondrial carrier proteins: one orthologous to the 2-oxoglutarate/malate carrier that imports malate into the organelle and exports oxoglutarate
out of the mitochondrial matrix, one orthologous to an
ADP/ATP carrier, and one carrier that does not specifically
cluster with any carrier of known specificity (PET8). The
oxoglutarate/malate carrier (Coll et al. 2003) also transports other dicarboxylates and tricarboxylates (such as oxaloacetate, malate, malonate, succinate, citrate, isocitrate,
cis-aconitate, and trans-aconitate). This carrier may also
play an important role in several metabolic functions requiring organic acid flux to or from the mitochondria, such
as nitrogen assimilation, transport of reducing equivalents
into the mitochondria, and fatty acid elongation (Picault
et al. 2002). Nyctotherus ovalis may use this carrier to import malate into the hydrogen-producing mitochondrion,
which is transformed into fumarate and used by Complex II
enzymes. In the sequenced genomes in supplementary table S1, Supplementary Material online, the oxoglutarate/
malate carrier is present in several mitochondria-bearing
species but absent from yeast, species with mitosomes,
and species with hydrogenosomes without an organellar
genome (supplementary table S1, Supplementary Material
online). The oxoglutarate/malate carrier and a putative
ADP/ATP carrier are also found in Blastocystis (Stechmann
et al. 2008).
Additional mitochondrial proteins that are lacking in
Trichomonas, and all species with mitosomes are MPV17
and ACN9, which have homologues in human and yeast
mitochondria (not shown in fig. 3). They are, however, present in Blastocystis. Consistent with its presence in N. ovalis,
MPV17 has been associated with mitochondrial genome
stability (Spinazzola et al. 2006), while ACN9 has been associated with respiration (Steinmetz et al. 2002).
Protein Import and Processing
We detected the mitochondrial import machinery components TOM 34, MAS 5, MPP, FtsH, peptidyl-prolyl cis-trans
isomerase, and protein disulfide isomerase. Hsps 10, 60, 70,
and 90 were also identified.
Mitochondrial Division
One gene, a dynamin-related protein, has been found that
might be engaged in mitochondrial division because it is an
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ortholog of the plant dynamin ADL2 that is known to be
involved in mitochondrial division (Arimura et al. 2004).
ROS Defense
To our surprise, we found three systems used to cope with
the detrimental effects of oxygen radicals. First of all, we
found thioredoxins that act as antioxidants by facilitating
the reduction of cysteines in other, oxidized proteins by
thiol-disulfide exchange. Second, of the glutathione system,
we found a glutathione reductase (EC 1.8.1.7) that reduces
glutathione disulfide (GSSG) to the sulfhydryl form GSH.
Finally, we found a peroxiredoxin.
The presence of enzymes supposed to react against oxygen radicals in an anaerobic environment might seem
strange, but Reactive oxygen species (ROS) can be produced
endogenously. Complex I is a major source of ROS, although
in mitochondria Complex III seems to be the principal site of
O2 (superoxide) formation (Chen et al. 2003).
HGT
To detect HGT, we followed the same procedure as previously described (Ricard et al. 2006). After clustering the sequences to remove redundancy, we determined the BH of
1,914 cDNAs and 2,841 gDNAs against a set of 165 complete proteomes. We identified 31 cDNAs and 25 gDNAs
with a bacterium as BH and 5 cDNAs and 6 gDNAs with an
archaeon as BH. We analyzed the 67 N. ovalis sequences
that have a bacterium or an archaeon as BH further, by
reconstructing the evolution of the proteins in a phylogenetic tree generated with PhyML. When requiring that the
N. ovalis sequence clustered with a high bootstrap value
with only noneukaryotic sequences, four genes could be
concluded to likely have been acquired through HGT (table
2; supplementary fig. S4a–d, Supplementary Material online).
These genes are an acetyl-CoA synthase (AMP-forming),
a b lactamase, a glycosylhydrolase, and an ornithine carbamoyltransferase. None of the proteins encoded by these
genes has however an N-terminal targeting signal. In contrast, for the [FeFe] hydrogenase evidence for HGT and organellar location have been documented before (Boxma
et al. 2007). This [FeFe] hydrogenase is remarkable because
it is fused with the 24 and 51 kD subunits of a bacterial
Complex I. This allows the use of NADH as electron donor.
That we found only one gene obtained by HGT (the hydrogenase) that unequivocally encodes an enzyme located
in the hydrogen-producing mitochondrion, suggests that
the role of HGT in the transformation to hydrogen-producing
mitochondrion is limited.
Conclusions
With the bioinformatic analysis of the organellar genome
and of the gDNAs and cDNAs, we are beginning to get
a comprehensive view of the metabolism of the hydrogen-producing mitochondrion of N. ovalis. First of all,
the organelle genome is a typical ciliate mitochondrial
genome, with the exception of the notable lack of components of Complexes III, IV, and V of the electron transport
chain. The latter feature is shared with the genome of the
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Table 2. Nyctotherus ovalis Genes That Likely Have Been Acquired by HGT From Bacteria/Archaea.
HGT (genus BH, id BH, sequence origin)
Acetyl-CoA synthetase
(Archaeoglobus, YP_003399995, gDNA incl telomeres)
b lactamase
(Pseudoalteromonas, YP_339054.1, gDNA incl telomere)
Glycosylhydrolase (glycosidase)
(Geobacillus, YP_003244808.1, gDNA)
Ornithine carbamoyltransferase
(Aeromonas, YP_858515.1, cDNA incl. polyA)
[FeFe] hydrogenase
(Spirochaeta, YP_003873858, gDNA, telomeres)
Accession Number
AJ871315
AM890088
AM894317
AM891292
AM890556
E.C. No Hsa Sce Tth Pte Pfa Cpa Tva Bla Pla Psp Ehi Gla Ecu
6.2.1.1 Y Y Y Y Y Y Y Y
N N Y
3.5.2.6 Y
N
Y
Y
N
Y
Y
N
N
N
N
3.2.1.-
N
N
N
N
N
N
N
N
N
N
N
AM896448
2.1.3.3 Y
Y
N
N
Y
N
Y
Y
N
Y
N
AY608627
1.12.7.2 N
N
N
N
N
N
Y
Y
Y
Y
N
Y
Y
NOTE.—The proteins associated with the genes were selected based on having a BH with Bacteria or Archaea, followed by a phylogenetic analysis using the 250 BHs in the
complete nonredundant database supplemented with the ten BHs in Eukaryotes and requiring a highly supported clustering of the N. ovalis gene with noneukaryotic genes,
see Materials and Methods for details. With each HGT candidate are indicated the genus name of the species with the BH, the National Center for Biotechnology
Information-identifier of the best hit, the source of the genetic material for the N. ovalis gene (cDNA or gDNA), and the extra evidence that the sequence is indeed derived
from N. ovalis (the presence of telomeres specific for N. ovalis genes or of a poly A tail for a cDNA). Y indicates the presence of an ortholog of the mitochondrial protein in
the corresponding species, N its absence. A blank cell is inserted if no answer is possible due to the incomplete genome. Species with mitochondria: (Hsa) Homo sapiens,
(Sce) Saccharomyces cerevisiae, (Tth) Tetrahymena thermophila, (Pte) Paramecium tetraurelia, (Ram) Reclinomonas americana, and (Pfa) Plasmodium falciparum; species
with hydrogenosomes: (Tva) Trichomonas vaginalis, (Bla) Blastocystis, (Pla) Psalteriomonas lanterna, and (Psp) Piromyces sp.; and species with a mitosomes: (Cpa)
Cryptosporidium parvum, (Ehi) Entamoeba histolytica, (Gla) Giardia lamblia, and (Ecu) Encephalitozoon cuniculi.
unrelated mitochondrion-like organelles of Blastocystis and
Proteromonas. This phenomenon is a striking example of
convergent evolution. Consequently, given the situation
with Nyctotherus, Blastocystis, and Proteromonas, we can
no longer use the production of ATP via oxidative phosphorylation as a defining feature of mitochondria, rather
what sets them apart from mitosomes and hydrogenosomes is the proton-pumping electron transport chain
of their mitochondrion-like organelles.
Moreover, the organelle of N. ovalis appears to host, besides
an incomplete TCA cycle, other ‘‘classical’’ mitochondrial
pathways, such as amino acid metabolism and fatty acid metabolism. A number of these proteins are typical for mitochondria and are not present in species with mitosomes
or hydrogenosomes without a genome. These include, of
course, proteins of the electron transport chain and of the
mitochondrial ribosome but also enzymes, such as succinylCoA synthetase (however present in Trichomonas), pyruvate
dehydrogenase, malate/a ketoglutarate antiporter, and 5,10methenyltetrahydrofolate synthetase (see also Ginger et al.
2010). What emerges is a metabolism that is in some aspects
(Complex III, Complex IV, and Complex V) reduced relative to
the metabolism of aerobic ciliate mitochondria and that is
strikingly similar to the metabolism of the mitochondrion-like
organelle of Blastocystis. However, N. ovalis also possesses
some extra proteins relative to aerobic ciliates. A few of these
appear to have been acquired by HGT. The case of the [FeFe]
hydrogenase is well documented (Boxma et al. 2007), but our
analysis has also unearthed a few others, like an acetyl-CoA
synthetase (AMP-forming). Nevertheless, their role in the adaptation to the anaerobic ecological niche of N. ovalis is not
obvious. Not all N. ovalis proteins that we found and that are
absent from the aerobic ciliates have been gained by HGT
from bacteria. We also observe a pathway for propionate metabolism that contains a propionyl CoA carboxylase and
a methylmalonyl CoA mutase. This pathway has not yet been
observed in ciliates, and besides its presence in metazoa, has
a patchy distribution among the eukaryotes, suggesting multiple loss events rather than HGT. Another example is a hybrid
cluster protein (prismane) that is absent from the sequenced
aerobic ciliates and that among the eukaryotes is mainly present in (faculatively) anaerobic species. However, the N. ovalis
sequence does not specifically cluster with bacterial sequences
(data not shown).
By definition, the organelle of N. ovalis is a hydrogenosome
because it generates hydrogen with the aid of an [FeFe] hydrogenase (Lindmark and Müller 1973). We have shown here
that this organelle is a mitochondrion that produces hydrogen, representing an intermediate state between mitochondria and genome-less hydrogenosomes. This allows us to
outline a hypothetical scheme for the evolution of hydrogenosomes. A possible scenario for eukaryotes with a textbook
mitochondrion genome and metabolism to evolve into an
organism with hydrogenosomes could be as follows (where
the order of events could be different):
1. Evolution of a reverse action of the TCA cycle (fumarate
respiration) as shown in a number of anaerobic mitochondria (Tielens et al. 2002; Tielens and van Hellemond
2007).
2. Recruitment of a hydrogenase like that shown in Naegleria
gruberi (Fritz-Laylin et al. 2010) and N. ovalis.
3. Loss of Complexes III, IV, and V of the electron transport
chain as shown in N. ovalis, Blastocystis, and Proteromonas.
4. Loss of the remaining part of the organellar genome as
shown, for example, in Trichomonas, Psalteriomonas, and
other organisms with hydrogenosomes.
It is clear that hydrogenosomes, hydrogen-producing
mitochondria, and mitochondrion-like organelles evolved
several times from different aerobic progenitors. The eukaryotic cell is clearly characterized by the presence of a
mitochondrion that has the capacity to adapt to anaerobic
environments by reductive evolution to yield hydrogenosomes (and mitosomes).
Supplementary Material
Supplementary table S1 and figures S1–S4 are available at
Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).
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Acknowledgments
The authors would like to thank Klaas Schotanus for his
help with the isolation of various N. ovalis sequences
and Michel van de Pol for his help in drawing some of
the figures. We also thank Prof. Alan Schwartz for revising
the English phrasing. G.D.M.v.d.S. and G.R. were supported
by the European Union fifth framework grant ‘‘CIMES’’
(QLK 3-2002-02151). I.S. was funded by the scholarship
SFRH/BD/32959/2006 from the Portuguese Foundation
for Science and Technology—FCT and by ‘‘Bolsas Rui Tavares 2010.’’ B.E.D. was funded by NWO HORIZON grant
050-71-058 and by NWO Veni grant 016.111.075. This work
was supported by the Netherlands Genomics Initiative
(Horizon Programme, project 050-71-555).
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